Project Summary/Abstract RNA editing and cleavage of chloroplast transcripts require proteins with the DYW-deaminase domain. The DYW-deaminase shares several features common to nucleotide deaminases and comprises the enzymatic portion of the RNA editing machinery. Some DYW-deaminase proteins are not associated with any RNA editing function and are required for ribonuclease cleavage. On this basis the domain can be broken down into one class with RNA editing functions and a second class with ribonuclease functions. Related enzymes function exclusively as amino hydrolases and are critical for various cellular functions including RNA editing, antibody diversity, and viral defense. Each DYW-deaminase domains binds two zinc ions. Zinc #1 is most likely coordinated by the typical C/HAE---CXXC motif found in all nucleotide deaminases. Amino acids in a unique C- terminus likely coordinate Zinc #2 and are nearby the catalytic zinc ion binding site. A general mechanism for cytidine deamination does not require two zinc ions. The second zinc ion could have a unique co-catalytic function, serve as a structural feature, or have a role in ligand binding. Zinc ions are not required for all ribonuclease mechanisms and addition of EDTA promotes the ribonuclease cleavage activity of recombinant DYW-deaminases in vitro. One or both zinc ions might operate as a zinc ?switch? that governs the deaminase and ribonuclease functions of the domain. This project aims to investigate a model where zinc ions act as a ?switch? that determines catalytic function. Zinc binding in DYW-deaminases with endoribonuclease and RNA editing functions will be compared to investigate if amino acid differences present in each functional classes determine zinc stoichiometry. A pool of recombinant DYW-deaminases with amino acid substitutions in putative zinc coordinating residues will be created to pinpoint the location of zinc coordinating residues. Endoribonuclease activities will be assayed for rDYW-deaminase proteins with known RNA editing functions as well as known endoribonuclease functions in the presence or absence of metal chelators. Ribonuclease activity will be measured for recombinant proteins with altered zinc stoichiometries and mutations in zinc coordinating residues. A series of proteins with amino acid changes might discover a link between zinc binding and enzymatic function as a well as to establish the reaction mechanism for RNA cleavage. An activity assay for RNA editing that includes addition of exogenous DYW-deaminases will be developed. The activity of DYW-deaminases with different zinc stiochiometries will be measured to determine the relationship between zinc ion binding and aminohydrolase activity. The second zinc ion may have a specialized role in ligand binding and to test this possibility the nucleic acid ligand requirements of the DYW-deaminase will be determined using RNAs and putative substrates with modified nucleotides. Finally, crystallization conditions for recombinant proteins will be screened. Crystals could be used in future experiments aimed at solving the structure of the DYW-deaminase.